Abstract: Membrane phospholipid biosynthesis and fatty acid metabolism are vital facets of bacterial physiology that are poorly understood. The long-term goal is to define the diversity of biochemical mechanisms used by bacterial pathogens to obtain lipid nutrients from the host, and to modify host unsaturated fatty acids to create new signaling molecules that alter the immune response. Fatty acid kinase is the only route for fatty acid activation in Gram-positive pathogens and we will establish whether this two-component system uses a phospho- cysteine intermediate in catalysis. X-ray crystallography and site-directed mutagenesis will define the structure and mechanism of this unique kinase. Staphylococcus aureus fatty acid kinase is essential for cellular lipid homeostasis and functions to re-cycle fatty acids into the phospholipid biosynthetic pathway. If fatty acids are allowed to accumulate, they inhibit the activity of SaeS, a master regulator of virulence factor transcription. We propose that cellular fatty acids arise from phospholipids, which serve as substrates in protein acylation and secondary metabolite synthesis. Characterizing these pathways will lead to the identification of the essential steps that enable the functionality of membrane lipoproteins and the virulence factor staphyloxanthin. A novel lipidomics workflow was developed to determine the structure of S. aureus membrane phospholipid at the infection site. This innovative approach will allow us to measure the extent of S. aureus utilization of host fatty acids for membrane formation, and determine the roles for the genes responsible for fatty acid acquisition. Streptococcus pneumoniae has a different lifestyle than S. aureus, and we will ascertain whether the number and substrate selectivities of the fatty acid binding proteins determine the differences in the way S. aureus and S. pneumoniae use host unsaturated fatty acids. This work is critical to understanding the mechanism of action of new antibiotics entering human clinical trials, like afabicin (Debiopharm), which are designed to target components of the bacterial fatty acid biosynthetic pathway. We discovered that S. aureus has an enzyme set that actively metabolizes host unsaturated fatty acids to create a spectrum of oxygenated products. We also found that S. aureus metabolizes prostaglandins. We will characterize the enzymes, transcriptional regulators and efflux pumps responsible for the production and release of these new biological effectors into the host environment. We will determine if these newly discovered pathways are a major countermeasure deployed by S. aureus to combat antimicrobial fatty acids produced by the innate immune system. We will also determine the structures of the S. aureus metabolites of host unsaturated fatty acids to verify that they are identical to gut metabolites that signal the immune system to create a more tolerant environment for the bacteria. Accomplishing these aims will define the importance of the acquisition and metabolism of host lipids for growth and virulence in S. aureus, validate the rationale for the use of antibiotics targeting of bacterial fatty acid synthesis, and serve as a blueprint for the understanding the physiology and virulence of other pathogens with similar gene sets.